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Development and validation of method for determination of clotrimazole in cream using HPLC
Poláčková, Jitka ; Matysová, Ludmila (advisor) ; Chocholoušová Havlíková, Lucie (referee)
The topic of my work was to optimalize and to validate the method for determination of capacity of the Clotrimazol, degradeted product (2-chlorophenyl) diphenylmethanol and conservations (methylparaben and propylparaben) in the preparation Clotrimazol ointment. My effort for this work was to develop the method that could half for total separation the particular defined substance in this sample, in the acceptable time, to find inside standard, to optimalize the conditions of separation and the method verify. The optimum chromatographic conditions were found on column Zorbax SB - Phenyl, 3,5 μm, 4,6 x 75 mm in the flow of mobile phase 0,5 ml per minute and wave - length 210 nm. Composition of the mobile phase was acetonitrile - water (55:45), pH water component was modified by acid phosphoric 5 % on 3,2. If these conditions are used, this method is validating. The testing parameters were accuracy, correctness, linearity, selectivity, robustness, detection and quantitative limit and test if the chromatographic system is acceptable.
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HPLC Determination of Lutein, Zeaxanthine and Betacarotene in Dietary Products
Dvořáková, Petra ; Šatínský, Dalibor (advisor) ; Chocholoušová Havlíková, Lucie (referee)
This rigorous thesis deals with modification of method for separation carotenoids lutein, betacarotene and zeaxanthin borrowed from diploma thesis (Optimization of chromatographic conditions for HPLC determination of chosen carotenoids, Petra Dvořáková, 2009). HPLC with VIS detection is the basic method. This method was modified by gradient elution for compressed time of analysis. Four ways of gradient elution were tested during optimization of new procedure. The second aim of this rigorous thesis is summary of extraction ways for carotenoids published in special literature and proposition of extraction procedures for carotenoids from dietary products. Extraction with organic solvents was used during searching for optimal way for extraction. Simple extraction with organic solvent was tested as basic method, but extraction with previous modification of sample (saponification or acidification of extraction medium) was tested too. In spite of testing of many various ways for extraction and many various organic solvents, there wasn't find optimal and universal extraction way.
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Determination of neopterin and its derivatives by HPLC with fluorescence detection.
Říhová, Lenka ; Chocholoušová Havlíková, Lucie (referee) ; Nováková, Lucie (advisor)
TITLE: Determination of neopterin and its derivates by HPLC with fluorescence detection SUMMARY: Neopterin, a pteridine derivative, is produced in human organism by monocytes or by B - lymphocytes stimulated with interferon γ or interleukine - 2. These active substances (interferon γ, interleukine - 2) are connected with immune system activation. Neopterin measurement in body fluids can therefore serve for monitoring of activation of various components of immune system induced by cytokines. Immune activation occurs for example during many infectious diseases, autoimmune diseases and malignant diseases. Recently the connection between neopterin and 7,8 - dihydroneopterin and intracellular oxidative stress and cell apoptosis was demonstrated in several scientific studies. Values of pteridines probably correlate with certain neurological disesases. Totally reduced form of biopterin serves as cofactor of some enzymes in human organism. In clinical practice the concentration of neopterin, eventually other related substances is expressed as the ratio neopterin / creatinine. This diploma thesis is dealing with the development of an analytical method for the determination of biologically active substances biopterin, 7,8 - dihydroneopterin, 5,6,7,8 - tetrahydroneopterin, neopterin and creatinine by high performance...
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The comparison of particle stationary phases with monolith in analysis of selected medicaments
Šestakova, Veronika ; Chocholoušová Havlíková, Lucie (referee) ; Matysová, Ludmila (advisor)
The performance of monolithic HPLC columns Chromolith® (made by Merck, Germany) and Onyx™ (made by Phenomenex, USA) and conventional C18 columns Discovery® (made by Supelco, Sigma-Aldrich, Prague, Czech Republic) and LiChroCart® 125-4 Purospher 5 µm (made by Merck, Germany) was tested and the comparison for two topical preparation Estrogel gel and Aphlox-K paste was made. The composition of mobile phase - for Estrogel gel analysis a mixture of acetonitrile, methanol and water (23:24:53, v/v/v) and for Aphlox-K paste analysis a mixture of methanol, water and acetic acid (50:50:1, v/v/v) - was usually not optimal for analysis at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the analyzed compounds. Various flow rates (0.3 - 5.0 ml/min) and mobile phase composition (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel analysis, following chromatographic conditions were found: using Chromolith® Flash RP18-e (25 mm x 4.6 mm), monolith column mobile phase was acetonitrile, methanol, water (16:24:60 v/v/v) and flow-rate 2.0 ml/min. Using monolith column Onyx™ Monolithic C18-e (50 mm x...
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Application of HPLC in pharmaceutical analysis.
Šestakova, Veronika ; Chocholoušová Havlíková, Lucie (referee) ; Matysová, Ludmila (advisor)
Determination of hexachlorophene in the product SEPTONEX ointment to the Department of Analytical Chemistry performed by HPLC. This method has many advantages over previously used titrate determination. The conditions of analysis are as follows: mobile phase is composed of 85% methanol with the addition of 1% acetic acid, column Purospher Merck RP C18, 5 mm (12.5 cm x 4 mm), flow rate of the mobile phase is 1.5 ml / min, sample injection volume is 20 µl. The aim was to optimize the use of methods, i.e. find the column using the modern trends in HPLC, which would allow obtaining precise, rapid and well reproducible results under similar chromatographic conditions. Based on the literature research were selected six different analytical columns with more or less similar characteristics as the column still used. In each of these columns have been measured solutions of standards hexachlorophene and solutions of ointment SEPTONEX samples at different flow rate of mobile phase. Other conditions of analysis have been retained. From the experiment showed that the tested columns is the most appropriate column Chromolith Performance RP18-e (10 cm x 4,6 mm) at a flow rate of mobile phase 2.0 ml / min. Another challenge was to find a suitable internal standard for faster and more accurate determination of...
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Determination of vitamine C and dehydroascorbic acid using UHPLC-MS method
Proroková, Zuzana ; Chocholoušová Havlíková, Lucie (referee) ; Nováková, Lucie (advisor)
A coupling of Ultra High Performance liquid chromatography with mass spectrometry provides a technique, which is rapid and sensitive. This thesis is focused on the use of UHPLC-MS for the determination of ascorbic acid (AA) and dehydroascorbic acid (DHA). AA is a small polar molecule that acts as an antioxidant. After oxidation AA creates DHA. AA/DHA ratio is an indicator of a redox state of organism. For the determination of AA and DHA several methods have been developed, which usually do not allow the simultaneous analysis, but require multistep subtraction procedure. The optimization of UHPLC-MS method for the determination of AA and DHA include the choice of mobile and stationary phase and mass spectrometry detector set- up. The choice of appropriate conditions depended mainly on retention time and the detector response. The effect of stationary phase, concentration, pH, composition of mobile phase on retention of AA and DHA was observed. Effect of mobile phase on stability of AA and DHA was observed as well. BEH Shield RP C18, BEH HILIC and BEH Amide column were compared. The best results were achieved on column BEH Shield RP C18. Measurements with 0.1%, 0.05%, 0.01% formic acid, 0.1%, 0.05%, 0.01% acetic acid, ammonium formate at pH 3.5 and amonium acetate at pH 4.4 and 6.8 as a water...
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Stanovení obsahu residuí kofeinu v povrchových vodách metodou HPLC s UV detekcí
Glosová, Ivana ; Solich, Petr (advisor) ; Chocholoušová Havlíková, Lucie (referee)
The occurence of caffeine residues was investigated in superrficial waters. The aim of this study was to obtain a first overview about contamination of waters from Mondego River and from source fontains near Coimbra city ( Portugal) with caffeine. The samples were preconcentrated by SPE ( Solid Phase Extraction) with Oasis polymeric column procedure and analysed by Liquid Chromatography (LC) with UV detektor ( 280nm). Mobile phase consist of 190 ml acetic acid glacial 100% and 810 ml of LC water (adjusted to pH 3.0 with acetate sodium) A total of twelve samples were collected in different site of Mondego River and from fountains near Coimbra city during three month's periods. A total of twelve samples of caffeine were analysed under the conditions described and none of them showed to be contaminated with caffeine.
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Dispersive Solid-Phase Extraction for the Analysis of Chosen Substances in Zingiber officinale
Urbanová, Martina ; Chocholoušová Havlíková, Lucie (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Martina Urbanová Consultant: PharmDr. Lucie Chocholoušová Havlíková, Ph.D. Thesis Title: Dispersive Solid-Phase Extraction for the Analysis of Chosen Substances in Zingiber officinale The thesis was focused on the development of optimal extraction method for the analysis of chosen substances contained in Zingiber officinale. Some representative samples containing extracts of Zingiber officinale were chosen. This method was based on the diploma thesis of Martina Urbanová [1]. To get a higher preconcentration factor, dispersive solid-phase extraction (dSPE) was tested for the sample pretreatment. Then, a method of supported liquid extraction (SLE) was examined as another methodology for the sample preparation. Chosen substances, 6-gingerol, 8-gingerol, 6-shogaol and 10-gingerol, were found in almost all samples. Among syrups, the largest amount of substances was contained in the Kitl syrup (preparation with cold water). Among teas, the highest number of substances was contained in Fruit-herbal tea flavored with ginger and lemon. 6-Shogaol was not found in rhizome samples, however, it has been found in the samples of teas and syrups. During the optimization of this method, various...
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